Dynophore-Based Approach in Virtual Screening: A Case of Human DNA Topoisomerase IIα

In this study, we utilized human DNA topoisomerase IIα as a model target to outline a dynophore-based approach to catalytic inhibitor design. Based on MD simulations of a known catalytic inhibitor and the native ATP ligand analog, AMP-PNP, we derived a joint dynophore model that supplements the static structure-based-pharmacophore information with a dynamic component. Subsequently, derived pharmacophore models were employed in a virtual screening campaign of a library of natural compounds. Experimental evaluation identified flavonoid compounds with promising topoisomerase IIα catalytic inhibition and binding studies confirmed interaction with the ATPase domain. We constructed a binding model through docking and extensively investigated it with molecular dynamics MD simulations, essential dynamics, and MM-GBSA free energy calculations, thus reconnecting the new results to the initial dynophore-based screening model. We not only demonstrate a new design strategy that incorporates a dynamic component of molecular recognition, but also highlight new derivates in the established flavonoid class of topoisomerase II inhibitors.

AK-I-190, a New Catalytic Inhibitor of Topoisomerase II with Anti-Proliferative and Pro-Apoptotic Activity on Androgen-Negative Prostate Cancer Cells

Castration-resistant prostate cancer (CRPC) is a clinical challenge in treatment because of its aggressive nature and resistance to androgen deprivation therapy. Topoisomerase II catalytic inhibitors have been suggested as a strategy to overcome these issues. We previously reported AK-I-190 as a novel topoisomerase II inhibitor. In this study, the mechanism of AK-I-190 was clarified using various types of spectroscopic and biological evaluations. AK-I-190 showed potent topoisomerase II inhibitory activity through intercalating into DNA without stabilizing the DNA-enzyme cleavage complex, resulting in significantly less DNA toxicity than etoposide, a clinically used topoisomerase II poison. AK-I-190 induced G1 arrest and effectively inhibited cell proliferation and colony formation in combination with paclitaxel in an androgen receptor–negative CRPC cell line. Our results confirmed that topoisomerase II catalytic inhibition inhibited proliferation and induced apoptosis of AR-independently growing prostate cancer cells. These findings indicate the clinical relevance of topoisomerase II catalytic inhibitors in androgen receptor-negative prostate cancer.

Establishing RNA-RNA interactions remodels lncRNA structure and promotes PRC2 activity

Human Polycomb Repressive Complex 2 (PRC2) catalysis of histone H3 lysine 27 methylation at certain loci depends on long noncoding RNAs (lncRNAs). Yet, in apparent contradiction, RNA is a potent catalytic inhibitor of PRC2. Here, we show that intermolecular RNA-RNA interactions between the lncRNA HOTAIR and its targets can relieve RNA inhibition of PRC2. RNA bridging is promoted by heterogeneous nuclear ribonucleoprotein B1, which uses multiple protein domains to bind HOTAIR regions via multivalent protein-RNA interactions. Chemical probing demonstrates that establishing RNA-RNA interactions changes HOTAIR structure. Genome-wide HOTAIR/PRC2 activity occurs at genes whose transcripts can make favorable RNA-RNA interactions with HOTAIR. We demonstrate that RNA-RNA matches of HOTAIR with target gene RNAs can relieve the inhibitory effect of a single lncRNA for PRC2 activity after B1 dissociation. Our work highlights an intrinsic switch that allows PRC2 activity in specific RNA contexts, which could explain how many lncRNAs work with PRC2.

LSD1 Promotes Prostate Cancer Cell Survival by Destabilizing FBXW7 at Post-Translational Level

Prostate cancer (PCa) is the most common cancer in men and the fifth leading cause of cancer death worldwide. Unfortunately, castration-resistant prostate cancer (CRPCa) is incurable with surgical treat and prone to drug resistance. Therefore, it is of great importance to find a new target for treatment. LSD1 is up-regulated in PCa and related with prognosis. The high-expression LSD1 has been shown to be a potential target for treatment and is widely studied for its demethylase-activity. However, its demethylation-independent function remains to be elusive in PCa. Recent study shows that LSD1 can destabilize cancer suppressor protein FBXW7 without demethylation-function. Hence, we hope to investigate the impact of non-canonical function of LSD1 on PCa cell survival. We over-expressed FBXW7 gene through plasmid vector in LNCaP and PC3 cell lines and the result shows that up-regulated FBXW7 can suppress the viability of PC cell through suppressing oncoproteins, such as c-MYC, NOTCH-1. After FBXW7 function experiment on PC cell, we knock-down LSD1 gene in the same kinds of cell lines. In western blot assay, we detected that down-regulation of LSD1 will cause the increasing of FBXW7 protein level and decreasing of its targeting oncoproteins. And mRNA level of FBXW7 did not change significantly after LSD1 knock-down, which means LSD1 may destabilize FBXW7 by protein-protein interactions. Moreover, exogenous wild type LSD1 and catalytically deficient mutant K661A both can abrogate previous effect of LSD1 knock-down. Consequently, LSD1 may promote PC cell survival by destabilizing FBXW7 without its demethylase-activity. Next, we compared two kinds inhibitors, and found that SP-2509 (Allosteric inhibitor) treatment suppress the cancer cell survival by blocking the LSD1–FBXW7 interaction, which is an effect that GSK-2879552 (catalytic inhibitor) cannot achieve. This work revealed a pivotal function of LSD1 in PCa, and indicated a new direction of LSD1 inhibitor research for PCa treatment.

DISCLOSING THE TRUE NATURE OF HESPERETIN’S ANTIGENOTOXICITY „IN VIVO“ WITHIN THE „DROSOPHILA MELANOGASTER“ SOMATIC CELLS THROUGH THE EXTENSIVE GENOTOXICAL AND STRUCTURE-BASED STUDIES

Previously unreported genotoxic and antigenotoxic potentials of hesperetin (Hes) were revealed by treating the Drosophila melanogaster (dm) whose DNA has been altered by means of O6-ethylguanine (dmGO6-Et) and O4-ethylthymine (dmTO4-Et) lesions appearance, caused by ethyl methanesulfonate (EMS), a proven alkylating agent and mutagen. Therefore, Hes potencies were determined by means of the comet assay on somatic cells level, where compound exerted no genotoxic effects but acted genotoxically as a Topoisomerase IIα (dmTopIIα) catalytic inhibitor by invading the Binding and Cleavage Domain and stabilizing the noncovalent dmTopIIα-plasmid DNA (dmPDNA) complex, as verified by the kinetoplast DNA (dmK-DNA) decatenation assays. Hes’s structure-based alignment caused compound’s A and C rings to occupy the area normally invaded by EMS, thus making a spatial barrier for the dmGO6-Et or dmTO4-Et lesions formation: the A ring C7-OH group formed hydrogen bonds (HBs) with either dmGO6 (dHB = 2.576 Å) or guanine’s N7 nitrogen (dmGN7, dHB = 2.737 Å), whereas the A ring C5-OH group formed an HB with dmTO4 (dHB = 3.548 Å). Furthermore, Hes likewise acted as a mixed-type competitive inhibitor of dmATPase, as verified by the catalytic, FRET, and structure-based studies where it affected the dmATPase dimerization and the hydrolysis of ATP, denying the metabolic energy for the catenation of ethylated G-dmDNA segment, the formation of dmTO4-Et-G-dmDNA phosphotyrosine intermediate (dmTO4-Et-G- dmDNA-PTyr785I), and the passage of ethylated T-dmDNA segment through the temporarily broken dmTO4-Et-G-dmDNA-PTyr785I, processes seen as comets. Conclusively, Hes may be used in anticancer therapy controlling the effects of alkylating agents.

Hybrid Inhibitors of DNA Gyrase A and B: Design, Synthesis and Evaluation

The discovery of multi-targeting ligands of bacterial enzymes is an important strategy to combat rapidly spreading antimicrobial resistance. Bacterial DNA gyrase and topoisomerase IV are validated targets for the development of antibiotics. They can be inhibited at their catalytic sites or at their ATP binding sites. Here we present the design of new hybrids between the catalytic inhibitor ciprofloxacin and ATP-competitive inhibitors that show low nanomolar inhibition of DNA gyrase and antibacterial activity against Gram-negative pathogens. The most potent hybrid 3a has MICs of 0.5 µg/mL against Klebsiella pneumoniae, 4 µg/mL against Enterobacter cloacae, and 2 µg/mL against Escherichia coli. In addition, inhibition of mutant E. coli strains shows that these hybrid inhibitors interact with both subunits of DNA gyrase (GyrA, GyrB), and that binding to both of these sites contributes to their antibacterial activity.